Protein Kinases
Protein kinases constitute a large family of structurally related enzymes that are responsible for the control of a wide variety of signal transduction processes within the cell (Hardie, G. and Hanks, S. (1995) The Protein Kinase Facts Book, I and II, Academic Press, San Diego, Calif.). The kinases may be categorized into families by the substrates they phosphorylate (e.g., protein-tyrosine, protein-serine/threonine, lipids, etc.). Sequence motifs have been identified that generally correspond to each of these kinase families (e.g., Hanks, S. K., Hunter, T., FASEB J., 9:576-596 (1995); Knighton, et al., Science, 253:407-414 (1991); Hiles, et al., Cell, 70:419-429 (1992); Kunz, et al., Cell, 73:585-596 (1993); Garcia-Bustos, et al., EMBO J., 13:2352-2361 (1994)).
Protein kinases may be characterized by their regulation mechanisms. These mechanisms include, for example, autophosphorylation, transphosphorylation by other kinases, protein-protein interactions, protein-lipid interactions, and protein-polynucleotide interactions. An individual protein kinase may be regulated by more than one mechanism.
Kinases regulate many different cell processes including, but not limited to, proliferation, differentiation, apoptosis, motility, transcription, translation and other signalling processes, by adding phosphate groups to target proteins. These phosphorylation events act as molecular on/off switches that can modulate or regulate the target protein biological function. Phosphorylation of target proteins occurs in response to a variety of extracellular signals (hormones, neurotransmitters, growth and differentiation factors, etc.), cell cycle events, environmental or nutritional stresses, etc. The appropriate protein kinase functions in signalling pathways to activate or inactivate (either directly or indirectly), for example, a metabolic enzyme, regulatory protein, receptor, cytoskeletal protein, ion channel or pump, or transcription factor. Uncontrolled signalling due to defective control of protein phosphorylation has been implicated in a number of diseases, including, for example, inflammation, cancer, allergy/asthma, diseases and conditions of the immune system, diseases and conditions of the central nervous system, and angiogenesis.
Apoptosis or programmed cell death is an important physiological process which removes cells no longer required by an organism. The process is important in early embryonic growth and development allowing the non-necrotic controlled breakdown, removal and recovery of cellular components. The removal of cells by apoptosis is also important in the maintenance of chromosomal and genomic integrity of growing cell populations. There are several known checkpoints in the cell growth cycle at which DNA damage and genomic integrity are carefully monitored. The response to the detection of anomalies at such checkpoints is to arrest the growth of such cells and initiate repair processes. If the damage or anomalies cannot be repaired then apoptosis is initiated by the damaged cell in order to prevent the propagation of faults and errors. Cancerous cells consistently contain numerous mutations, errors or rearrangements in their chromosomal DNA. It is widely believed that this occurs in part because the majority of tumours have a defect in one or more of the processes responsible for initiation of the apoptotic process. Normal control mechanisms cannot kill the cancerous cells and the chromosomal or DNA coding errors continue to be propagated. As a consequence restoring these pro-apoptotic signals or suppressing unregulated survival signals is an attractive means of treating cancer.
The signal transduction pathway containing the enzymes phosphatidylinositol 3-kinase (PI3K), PDK1 and PKB amongst others, has long been known to mediate increased resistance to apoptosis or survival responses in many cells. There is a substantial amount of data to indicate that this pathway is an important survival pathway used by many growth factors to suppress apoptosis. The enzyme PI3K is activated by a range of growth and survival factors e.g. EGF, PDGF and through the generation of polyphosphatidylinositols, initiates the activation of the downstream signalling events including the activity of the kinases PDK1 and protein kinase B (PKB) also known as Akt. This is also true in host tissues, e.g. vascular endothelial cells as well as neoplasias.
Protein Kinase p70S6K
The 70 kDa ribosomal protein kinase p70S6K (also known as SK6, p70/p85 S6 kinase, p70/p85 ribosomal S6 kinase and pp70s6k) is a member of the AGC subfamily of protein kinases. p70S6K is a serine-threonine kinase that is a component of the phosphatidylinositol 3 kinase (PI3K)/AKT pathway. p70S6K is downstream of PI3K, and activation occurs through phosphorylation at a number of sites in response to numerous mitogens, hormones and growth factors. This response may be under the control of mTOR since rapamycin acts to inhibit p70S6K activity and blocks protein synthesis, specifically as a result of a down-regulation of translation of these mRNA's encoding ribosomal proteins. p70S6K is also regulated by PI3K and its downstream target AKT. Wortmannin and rapamycin cause a decrease in p70S6K phosphorylation at sites dependent of the PI3K pathway. Mutant p70S6K is inhibited by wortmannin but not by rapamycin suggesting that the PI3K pathway can exhibit effects on p70S6K independent of the regulation of mTOR activity.
The enzyme p70S6K modulates protein synthesis by phosphorylation of the S6 ribosomal protein. S6 phosphorylation correlates with increased translation of mRNAs encoding components of the translational apparatus, including ribosomal proteins and translational elongation factors whose increased expression is essential for cell growth and proliferation. These mRNAs contain an oligopyrimidime tract at their 5′ transcriptional start (termed 5′TOP), which has been shown to be essential for their regulation at the translational level.
In addition to its involvement in translation, p70S6K activation has also been implicated in cell cycle control, neuronal cell differentiation, regulation of cell motility and a cellular response that is important in tumor metastases, the immune response and tissue repair. Antibodies to p70S6K abolish the mitogenic response driven entry of rat fibroblasts into S phase, indication that p70S6K function is essential for the progression from G1 to S phase in the cell cycle. Furthermore inhibition of cell cycle proliferation at the G1 to S phase of the cell cycle by rapamycin has been identified as a consequence of inhibition of the production of the hyperphosphorylated, activated form of p70S6K.
The tumor suppressor LKB1 activates AMPK which phosphorylates the TSC1/2 complex in the mTOR/p70S6K pathway, therefore feeds into p70S6K through a PKB independent pathway. Mutations in LKB1 cause Peutz-Jeghers syndrome (PJS), where patients with PJS are 15 times more likely to develop cancer than the general population. In addition, ⅓ of lung adenocarcinomas harbor inactivating LKB1 mutations.
A role for p70S6K in tumor cell proliferation and protection of cells from apoptosis is supported based on its participation in growth factor receptor signal transduction, overexpression and activation in tumor tissues. For example, Northern and Western analyses revealed that amplification of the PS6K gene was accompanied by corresponding increases in mRNA and protein expression, respectively (Cancer Res. (1999) 59: 1408-11—Localization of PS6K to Chromosomal Region 17q23 and Determination of Its Amplification in Breast Cancer).
Chromosome 17q23 is amplified in up to 20% of primary breast tumors, in 87% of breast tumors containing BRCA2 mutations and in 50% of tumors containing BRCA1 mutations, as well as other cancer types such as pancreatic, bladder and neuroblastoma (see M Barlund, O Monni, J Kononen, R Cornelison, J Torhorst, G Sauter, O-P Kallioniemi and Kallioniemi A, Cancer Res., 2000, 60:5340-5346). It has been shown that 17q23 amplifications in breast cancer involve the PAT1, RAD51C, PS6K, and SIGMA1B genes (Cancer Res. (2000): 60, pp. 5371-5375).
The p70S6K gene has been identified as a target of amplification and overexpression in this region, and statistically significant association between amplification and poor prognosis has been observed.
Clinical inhibition of p70S6K activation was observed in renal carcinoma patients treated with CCI-779 (rapamycin ester), an inhibitor of the upstream kinase mTOR. A significant linear association between disease progression and inhibition of p70S6K activity was reported.
p70S6K has been implicated in metabolic diseases and disorders. It was reported that the absence of p70S6 protects against age- and diet-induced obesity while enhancing insulin sensitivity. A role for p70S6K in metabolic diseases and disorders such as obesity, diabetes, metabolic syndrome, insulin resistance, hyperglycemia, hyperaminoacidemia, and hyperlipidemia is supported based upon the findings.
ROCK Kinases
The ROCK kinase family comprises two known members: ROCK1 and ROCK2:                ROCK1. Synonyms: Rho-associated protein kinase 1; p160 ROCK; P160 ROK; p160 ROCK-1, Rho-associated, coiled-coil containing protein kinase 1; Rho kinase 1; ROK beta.        ROCK2. Synonyms: Rho-associated protein kinase 2; p164 ROCK; p164 ROK; p164 ROCK-2; Rho-associated, coiled-coil containing protein kinase 2, Rho kinase 2; ROK alpha.        
The process of metastasis involves a restructuring of the cytoskeleton as well as cell-cell and cell-matrix adhesions allowing cells to break away from the tumor mass, invade local tissue, and ultimately spread throughout the body. These effects on cell morphology and adhesion are regulated by members of the Rho GTPase family.
Activated RhoA is capable of interacting with several effector proteins including the ROCK kinases ROCK1 and ROCK2. ROCK1 and ROCK2 can be activated by the RhoA-GTP complex via physical association. Activated ROCKs phosphorylate a number of substrates and play important roles in pivotal cellular functions. The substrates for ROCKs include myosin binding subunit of myosin light chain phosphatase (MBS, also named MYPT1), adducin, moesin, myosin light chain (MLC), LIM kinase, and the transcription factor FHL. The phosphorylation of theses substrates modulate the biological activity of the proteins and provide a means to alter a cell's response to external stimuli.
Elevated expression of RhoA and RhoC, as well as the Rho effector proteins ROCK1 and ROCK2, are commonly observed in human cancers, including in the progression of testicular germ cell tumours, small breast carcinomas with metastatic ability, invasion and metastasis of bladder cancer, tumor progression in ovarian carcinoma.
Progression of tumors to invasive and metastatic forms requires that tumor cells undergo dramatic morphologic changes, a process regulated by Rho GTPases. Actomyosin contractility is a mechanism by which cells exert locomotory force against their environment. Signalling downstream of the small GTPase Rho increases contractility through ROCK-mediated regulation of myosin-II light chain (MLC2) phosphorylation.
The ROCK kinases are thought to participate in the induction of focal adhesions and stress fibers and to mediate calcium sensitization of smooth muscle contraction by enhancing phosphorylation of the regulatory light chain of myosin.
In vivo studies have also shown that ROCK inhibition reduced the invasiveness of several tumor cell lines. ROCK inhibitors, such as Y-27632 or WF-536, have been used in some studies to demonstrate these properties.
Inhibitors of ROCKs have been suggested for use in the treatments of a variety of diseases. These include cardiovascular diseases such as hypertension, chronic and congestive heart failure, cardiac hypertrophy, restenosis, chronic renal failure and atherosclerosis. Also, because of its muscle relaxing properties, inhibitors may also be suitable for asthma, male erectile dysfunction, female sexual dysfunction and over-active bladder I syndrome.
ROCK inhibitors have been shown to possess anti-inflammatory properties. Thus they can be used as treatment for neuroinflammatory diseases such as stroke, multiple sclerosis, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and inflammatory pain, as well as other inflammatory diseases such as rheumatoid arthritis, irritable bowel syndrome, and inflammatory bowel disease. Based on their neurite outgrowth inducing effects, ROCK inhibitors could be useful drugs for neuronal regeneration, inducing new axonal growth and axonal rewiring across lesions within the CNS. ROCK inhibitors are therefore likely to be useful for regenerative treatment of CNS disorders such as spinal cord injury, acute neuronal injury (stroke, traumatic brain injury), Parkinsons disease, Alzheimers disease and other neurodegenerative disorders. Since ROCK inhibitors reduce cell proliferation and cell migration, they could be useful in treating cancer and tumor metastasis. Finally, there is evidence to suggest that ROCK inhibitors suppress cytoskeletal rearrangement upon virus invasion, thus they also have potential therapeutic value in anti-viral and anti-bacterial applications. ROCK inhibitors are also useful for the treatment of insulin resistance and diabetes.
ROCK Inhibitor Y-27632
Adhesion of tumour cells to host cell layers and subsequent transcellular migration are pivotal steps in cancer invasion and metastasis. The small GTPase Rho controls cell adhesion and motility through reorganization of the actin cytoskeleton and regulation of actomyosin contractility. Cultured rat MM1 hepatoma cells migrate in a serum-dependent, Rho-mediated manner, through a mesothelial cell monolayer in vitro. Among several proteins isolated as putative target molecules of Rho, the ROCK kinases are thought to participate in the induction of focal adhesions and stress fibres in cultured cells, and to mediate calcium sensitization of smooth muscle contraction by enhancing phosphorylation of the regulatory light chain of myosin. Transfection of MM1 cells with cDNA encoding a dominant active mutant of ROCK conferred invasive activity independently of serum and Rho. In contrast, expression of a dominant negative, kinase-defective ROCK mutant substantially attenuated the invasive phenotype.
A specific ROCK inhibitor (Y-27632) blocked both Rho-mediated activation of actomyosin and invasive activity of these cells. Furthermore, continuous delivery of this inhibitor using osmotic pumps considerably reduced the dissemination of MM1 cells implanted into the peritoneal cavity of syngeneic rats. These results indicate that ROCK plays an essential part in tumor cell invasion, and demonstrate its potential as a therapeutic target for the prevention of cancer invasion and metastasis.
VEGF induced the activation of RhoA and recruited RhoA to the cell membrane of human ECs. This increase in RhoA activity is necessary for the VEGF-induced reorganization of the F-actin cytoskeleton, as demonstrated by adenoviral transfection of dominant-negative RhoA. Rho kinase mediated this effect of RhoA, as was demonstrated by the use of Y-27632, a specific inhibitor of Rho kinase. Inhibition of Rho kinase prevented the VEGF-enhanced EC migration in response to mechanical wounding but had no effect on basal EC migration. Furthermore, in an in vitro model for angiogenesis, inhibition of either RhoA or Rho kinase attenuated the VEGF-mediated ingrowth of ECs in a 3-dimensional fibrin matrix. CONCLUSIONS: VEGF-induced cytoskeletal changes in ECs require RhoA and Rho kinase, and activation of RhoA/Rho kinase signaling is involved in the VEGF-induced in vitro EC migration and angiogenesis.
Y-27632 can relax smooth muscle and increase vascular blood flow. Y-27632 is a small molecule that can enter cells and is not toxic in rats after oral administration of 30 mg/kg for 10 days. Effective doses for the use of this compound are approximately 30 μM. It reduces blood pressure in hypertensive rats, but does not affect blood pressure in normal rats. This has led to the identification of Rho signalling antagonists in treatment of hypertension (Somlyo, 1997 Nature 389:908; Uehata et al., 1997 Nature 389:990).
The use of a specific inhibitor of ROCK, Y-27632 (Uehata, et al., Nature, 389, 990 994, 1997, Davies, et al., Biochemical Journal., 351, 95-105, 2000, and Ishizaki, et al., Molecular Pharmacology., 57, 976-983, 2000), has demonstrated a role for this enzyme in Ca2+ independent regulation of contraction in a number of tissues, including vascular (Uehata, et al., Nature., 389, 990-994, 1997), airway (Ilikuka et al., European Journal of 30 Pharmacology., 406, 273-279, 2000) and genital (Chitaley at al., Nature Medicine., 7(1), 119-122, 2001) smooth muscles. In addition, Jezior et al. British Journal of Pharmacology., 134, 78-87, 2001 have shown that Y-27632 attenuates bethanechol-evoked contractions in isolated rabbit urinary 35 bladder smooth muscle.
The Rho kinase inhibitor Y-27632 has been tested for the following disease applications:                Hypertension (Uehata et al., 1997 IBID; Chitaley at al., 2001a IBID; Chrissobolis and 15 Sobey, 2001 C. Circ. Res 88:774)        Asthma (Iizuka et al., 2000 Eur. J. Pharmacol 406:273; Nakahara et al. Eur. J. Pharmacol 389:103, 2000)        Pulmonary vasoconstriction (Takamura et al., 2001 Hepatology 33:577)        Vascular disease (Miyata et al., 2000 Thromb Vasc Biol 20:2351; Robertson et al., 2000 Br. J. Pharmacol 131:5)        Penile erectile dysfunction (Chitaley et al., 2001b Nature Medicine 7:119; Mills et al., 2001 J. Appl. Physiol. 91: 1269; Rees et al., Br. J. Pharmacol 133:455 2001)        Glaucoma (Honjo et al., 2001 Methods Enzymol 42:137; Rao et al., 2001 Invest. Opthalmol. Urs. Sci. 42:1029)        Cell transformation (Sahai et al., 1999 Curr. Biol. 9:136-5)        Prostate cancer metastasis (Somlyo et al., 2000 BBRC 269:652)        Hepatocellular carcinoma and metastasis (Imamura et al., 2000; Takamura et al., 2001)        Liver fibrosis (Tada et al., 2001 J. Hepatol 34:529; Wang et al., 2001 Am. J. Respir. Cell Mol Biol. 25:628)        Kidney fibrosis (Ohlci et al., J. Heart Lung Transplant 20:956 2001)        Cardioprotection and allograft survival (Ohlci et al., 2001 IBID)        Cerebral vasospasm (Sato et al., 2000 Circ. Res 87: 195).ROCK Kinase and Cardiovascular Disease        
There is growing evidence that ROCKs, the immediate downstream targets of the small guanosine triphosphate-binding protein Rho, may contribute to cardiovascular disease. ROCKs play a central role in diverse cellular functions such as smooth muscle contraction, stress fiber formation and cell migration and proliferation. Overactivity of ROCKs is observed in cerebral ischemia, coronary vasospasm, hypertension, vascular inflammation, arteriosclerosis and atherosclerosis. ROCKs, therefore, may be an important and still relatively unexplored therapeutic target in cardiovascular disease. Recent experimental and clinical studies using ROCK inhibitors such as Y-27632 and fasudil have revealed a critical role of ROCKs in embryonic development, inflammation and oncogenesis. This review will focus on the potential role of ROCKs in cellular functions and discuss the prospects of ROCK inhibitors as emerging therapy for cardiovascular diseases.
Abnormal smooth-muscle contractility may be a major cause of disease states such as hypertension, and a smooth-muscle relaxant that modulates this process would be useful therapeutically. Smooth-muscle contraction is regulated by the cytosolic Ca2+ concentration and by the Ca2+ sensitivity of myofilaments: the former activates myosin light-chain kinase and the latter is achieved partly by inhibition of myosin phosphatase. Rho signaling pathways in vascular smooth muscle cells are highly activated in hypertension, a condition associated with a variety of vascular diseases, including restenosis injury and atherosclerosis.
Hypertension is a cardiovascular disorder characterized by increased peripheral vascular resistance and/or vascular structural remodeling. Recently, rapidly growing evidence from hypertensive animal models suggests that small GTPase Rho and its downstream effector, Rho-kinase, play an important role in the pathogenesis of hypertension. Activation of the Rho/Rho-kinase pathway is essential for smooth muscle contractility in hypertension. A greater RhoA expression and an enhanced RhoA activity have been observed in aortas of hypertensive rats, such as genetic spontaneously hypertensive rats and N(omega)-nitro-L-arginine methyl ester-induced hypertension.
ROCK Kinase and Neurological Diseases
Abnormal activation of the Rho/ROCK pathway has been observed in various disorders of the central nervous system. Injury to the adult vertebrate brain and spinal cord activates ROCKs, thereby inhibiting neurite growth and sprouting. Inhibition of ROCKs results in accelerated regeneration and enhanced functional recovery after spinal-cord injury in mammals, and inhibition of the Rho/ROCK pathway has also proved to be efficacious in animal models of stroke, inflammatory and demyelinating diseases, Alzheimer's disease and neuropathic pain. ROCK inhibitors therefore have potential for preventing neurodegeneration and stimulating neuroregeneration in various neurological disorders.
The development of a neuron requires a series of steps that begins with migration from its birth place and initiation of process outgrowth, and ultimately leads to differentiation and the formation of connections that allow it to communicate with appropriate targets. Over the past several years, it has become clear that the Rho family of GTPases and related molecules play an important role in various aspects of neuronal development, including neurite outgrowth and differentiation, axon pathfinding, and dendritic spine formation and maintenance.
One common denominator for both neurite outgrowth inhibition and neurite repulsion is actin rearrangements within the growth cone. Central to the regulation of the actin cytoskeleton in both neuronal and non-neuronal cells is the Rho family of small GTPases. Rho family members cycle between an inactive GDP-bound form and an active GTP-bound form. Several lines of evidence suggest that manipulating the activity state of Rho GTPases may modulate growth cone collapse and neurite outgrowth inhibition.
More recently, behaviorally, inactivation of Rho pathway can induce rapid recovery of locomotion and progressive recuperation of forelimb-hindlimb coordination. These findings provide evidence that the Rho signaling pathway is a potential target for therapeutic interventions after spinal cord injury.